Preeclampsia (PE) is a serious complication of pregnancy manifested by high blood pressure, proteinuria, and edema, sometimes with encephalopathy, seizures, and hepatic failure. PE complicates from 5 to 10% of pregnancies, and is a major cause of maternal a fetal morbidity and mortality worldwide. Elevation in the circulating level of an endogenous digoxin-like factor (EDLF), an unknown substance that cross reacts with anti-digoxin antibodies and inhibits the NA+/K+ ATPase (NKA) was first noted in the 1980s. An extensive literature supports the hypothesis that increased levels of EDLF may be causative factor in the pathogenesis of hypertension. Recently, marinobufagenin (MBG), an endogenous cardiotonic steroid (CTS), has been identified as the EDLF. Plasma MBG is elevated over five-fold in pregnancies complicated by PE, suggesting a role pathophysiological role. Digibind, an anti-digoxin antiserum that cross-reacts MBG, provided significant in improvement in renal function relative to placebo among 51 patients with severe PE, with no adverse effects. Monoclonal antibodies to MBG are in clinical trials for PE. The lack of commercially available assays severely hampers research on the role MBG and at least two other cardiotonic steroids in PE and other diseases. The overall goal of this project is to develop an immunoassay panel for MBG and other potentially important endogenous cardiotonic steroids as a research tool and, ultimately, a clinical diagnostic for MBG levels in plasma and urine. In Phase I, we will identify monoclonal antibodies specific for each cardiotonic steroid demonstrating sensitivity and specificity. We will configure a competitive ELISA and will optimize it for the quantitation each CTS in plasma and urine. Finally, we will translate our competitive ELISA to a novel, automated immunoassay platform with ultrasensitive electrochemical detection. Phase II work will obtain data necessary for the submission of a 510k to support future commercialization.